The number of biologically active peptides is quite large. However, their potential utility as response modifiers, neuroeffectors or immunomodulators is dramatically circumscribed by their demonstrated very short half-lives in vivo and their lack of effectiveness when administered orally. This latter phenomenon is primarily due to the extreme ability of biologically active polypeptides in the presence of the peptidases and proteases normally found in the digestive tract.
It is desirable to stabilize the backbone amide linkages of these biologically active peptides against such proteolytic enzymes in order to improve the pharmacokinetic properties of these peptides. Enhanced stability to enzymatic degradation would make these peptides more useful therapeutic agents.
Recent advances in chemical replacement or modification of peptide linkages indicate that such linkage stabilization is feasible. By replacement of peptide linkages with thioamide bonds at those positions of the peptide backbone responsible for the biological response-limiting cleavage by peptidases and proteases, an increased stability to enzymatic degradation is obtained for many thiopeptide analogs. Reid and von Der Emden (W. Reid and W. von Der Emden, "Aminosaure-thionester und Endothiopeptide, II", Liebigs Ann. Chem., 642, 128, (1961)) discuss racemic thioamide formation through the thionester thioacylating agent: ##STR2## wherein R and R' are selected from lower alkyl and aryl. Further, enhanced pharmacological activity is exhibited for many of these analogues. Lajoie, et al., (G. Lajoie, F. Lepine, S. LeMaire, F. Jolicoeur, C. Aube, A. Turcotte and B. Belleau, "Synthesis and Biological Activity of Monothionated Analogs of Leucine-enkephalin", Int. J. Pept. Protein Res., 24, 316, (1984)). Thiopeptide derivatives have demonstrated increased activity in vivo as biological response modifiers, neuroeffectors, and immunomodulators as compared with their oxygenated analogs. For example, Causen, et al. (K. Clausen, A. Spatola, C. Lemieux, P. Schiller, and S. Lawesson, "Evidence of a Peptide Backbone Contribution Toward Selective Receptor Recognition for Leucine Enkephalin Thioamide Analogs", Biochem. Biophys. Res. Commun., 120, 305, (1984)) demonstrate the increased pharmacological activity of one such thiopeptide analog over its oxygenated counterpart.
Methods for replacement for the carbonyl oxygen atom of a carboxyl moiety with a sulphur atom are known. Clausen, et al. (K. Clausen, M. Thorsen, and S. Lawesson, "Studies on Amino Acids and Peptides. Part 6. Methods for Introducing Thioamide Bonds into the Peptide Backbone: Synthesis of the Four Monothio Analogues of Leucine Enkephalin", J. Chem. Soc. Perkin Trans., 785, (1984)) describe thioacylation by the use of dithioesters of the formula: ##STR3## wherein Z is carbobenzoxy and R is selected from hydrogen, lower alkyl, and aryl. No information, however, regarding racemization is described. It is also known that the thiopeptides so formed are useful reagents and intermediates for further thiopeptide synthesis. See, P. Campbell, and N. Nashed, "Carboxypeptidase A Catalyzed Hydrolysis of Thiopeptide and Thionester Analogues of Specific Substrates. An Effect on K.sub.cat for Peptide, but not Ester, Substrates", J. Am. Chem. Soc., 104, 5221-26, (1982); P. Bartlett, K. Speer, and N. Jacobsen, "A Thioamide Substrate of Carboxypeptidase A", Biochemistry, 21, 1608-11, (1982); and L. Maziak, G. Lajoie, and B. Belleau, "Productive Conformation in the Bound State and Hydrolytic Behavior of Thiopeptide Analogues of Angiotensin-converting Enzyme Substrates", J. Am. Chem. Soc., 108, 182-83, (1986). Such thiopeptide derivatives also have shown resistance to enzymatic hydrolysis. W. Reid and E. Schmidt, "N-acylierte .alpha.-Aminoimidosaureester, Imidodipeptide and Endothiodipeptide", Liebigs Ann. Chem., 695, 217, (1966), for example, disclose the synthesis of a protected amino acid thionester as an intermediate in the preparation of a thiopeptide in moderate yield.
Thionation of peptides, or the replacement of an oxygen atom with a sulphur atom, at the carbonyl functionality of their peptide bonds has heretofore demonstrated a lack of reaction site specificity. Decreased overall yields have been observed because of side reactions and the by-products so formed which cause the purity of the product and the efficiency of the reaction to suffer. Further, the optical integrity of the final product is often not maintained due to the reaction mechanism of the previously used thioacylating reagents. The limited effectiveness of these thioacylating reagents severely circumscribed the potential of thiopeptides as pharmacological agents. Lack of an efficient method of producing pure, optically active thiopeptides has rendered the evaluation of pharmacological activity, stability to enzymatic and pH degradation, and toxicity of such compounds very difficult, since sufficient quantities of these materials have heretofore been unobtainable.
The optical integrity of a compound relates to its ability to rotate light. This ability is measured in an instrument known as a polarimeter which utilizes a zero point reference. The degree to which a chemically pure material rotates light indicates its relative optical purity. That is, a material may be chemically pure while being optically inactive or racemic. The amount of activity that is observed from a material is often dependent upon its optical purity. Two enantiomers although possessing idential chemical formulae may have completely different biological activities. It is common in medicinal applications for a compound of one optical configuration to exhibit activity and usefulness, while its optical rotamer or complementary enantiomer demonstrates a different activity or is wholly inert. Thus, where optical configuration is important, optical purity, as well as chemical purity, is an important concern.
It is desirable that a thiopeptide meet several criteria to be suitable for pharmacological study. First, the thiopeptide should demonstrate an increased resistance to enzymatic degradation. Second, the thiopeptide should elicit an enhanced biological response over its oxygenated counterpart. Third, it must be safe for human ingestion. Fourth, the thiopeptide should be capable of being produced in quantities large enough to perform clinical studies.
Regardint he first three concerns, the characteristics described should be possessed as inherent properties of the thiopeptide which establish it as superior to other peptides not containing a thioamide linkage between adjacent amino acid residues. With reference to the last criterion, it is advantageous to be capable of producing large quantities of material. Several factors are important with respect to this consideration. The process for producing the thiopeptide is preferably simple, efficient and economical. That is, the reaction scheme of the process should contain few steps, afford high overall yields, and demonstrate minimal by-product formation. Moreover, the scheme should preferably utilize inexpensive reagents and materials. Further, the method should ensure the optical integrity of the growing peptide by avoiding reations that will reacemize the compound. That is, a racemic mixture is likely not to fully exhibit the desired pharmacological response.
Prior thioacylation processes have suffered from being cumbersome and complicated, Moreover, they do not afford products with a high degree of optical integrity and provide inadequate overall yields of the thiopeptide.
Accordingly, there is a need for thioacyclating reagents which permit the selective incorporation of thioamide linkages into growing peptides at specific residue linkages while utilizing efficient reaction conditions. There is also a need for a thioacylation process which will retain the optical integrity of the resulting peptide and will produce such peptide in high yields. There is additionally a need for methods for preparing thioacylating reagents capable of simple and economical reaction with amino acids and peptides to produce thiopeptides. There is yet another need for thiopeptides, and methods to prepare them, having increased enzymatic stability and enhanced biological activity over their oxygenated analogs.